Factors governing bone marrow adipose tissue (BMAT) are not yet well-defined, despite the great importance of this adipose depot in health and disease. The goal of this project is to test our hypothesis that osteocytes communicate with and regulate adipogenic progenitor cells to modulate BMAT through secretion of the soluble mediator sclerostin. In osteoporosis, obesity, and normal aging, BMAT volume is increased, and BMAT may be an independent risk factor for fracture. High BMAT also leads to disrupted hematopoiesis, altered immune function, reduced bone mineral density, and may increase the risk of bone metastasis. Preliminary data demonstrate a novel ability for sclerostin to stimulate adipogenesis, supporting a paradigm-shifting notion that sclerostin, potentially through inhibition of the Wnt signaling pathway, may be a pro-adipogenic, as well as an anti-osteogenic factor. In Aim 1, the influence of osteocytes, and their secreted osteokine sclerostin, on adipogenesis of bone marrow pre- adipocytes will be determined in vitro with direct or indirect co-cultures, in both 2D and novel 3D co- culture systems. These will be analyzed with confocal microscopy imaging, lipidomic analysis, gene and protein expression, and histology. The signaling pathway of sclerostin within the adipocyte will be elucidated, which we hypothesize acts via Wnt inhibition to prevent beta-catenin activation. In Aim 2, the influence of osteocytes and sclerostin on adipogenesis will be determined in vivo. Complementary mouse models will be utilized to characterize BMAT in sclerostin knockout (elevated bone) and osteocalcin-cre diphtheria toxin receptor mice (decreased bone) models and determine the effects of diet, body mass index, and sclerostin on this phenotype. Assessments will be done on BMAT (content, lipid profile, phenotype, and gene/protein expression), bone phenotype (CT, histology), whole body composition, and circulating blood serum. These studies translate to the clinic by identifying key elements in the pathophysiology of disorders such as osteoporosis and obesity. The results could profoundly alter our current understanding of the bone marrow niche in health and disease, enabling the development of more effective therapies. In summary, the long-term objective of this project is to test the novel hypothesis that osteocytes, via sclerostin or other molecules, provide a unidirectional stimulus for marrow adipogenesis. We will test this hypothesis in novel 3D in vitro systems and repurposed in vivo models to identify the molecular mechanisms and signaling pathways governing this process.